1.Effects of nuclear translocation of tissue transglutaminase and the release of cytochrome C on hepatocyte apoptosis.
Liangwen SONG ; Xianmei MA ; Yang LI ; Xuemei CUI ; Xiaomin WANG
Chinese Medical Journal 2003;116(5):742-746
OBJECTIVETo assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes.
METHODSHepatocytes isolated from tissue transglutaminase gene knock-out mice and rats were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by (14)C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.
RESULTSTTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.
CONCLUSIONSIncreased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.
Animals ; Apoptosis ; Cell Nucleus ; metabolism ; Cytochrome c Group ; metabolism ; Hepatocytes ; cytology ; metabolism ; Male ; Mice ; Rats ; Signal Transduction ; Transglutaminases ; metabolism
2.Mitochondrial apoptotic signaling pathway in neurons following brain injury induced by hypoxia.
Kui-ming ZHANG ; Zhi-gang LIAO
Journal of Forensic Medicine 2004;20(3):178-182
Impairment of neuronal mitochondria following hypoxia of brain not only result in nerve cell's energy-deprivation and dysfunction, mitochondria also play key roles in apoptosis of neurons. A central step being the release of cytochrome c (cyt c) across the outer mitochondrial membrane into the cytoplasm through opening of the mitochondrial permeability transition pore. Releasing of cytochrome c induce to downstream consequences of specific caspase activation. The antiapoptotic and proapoptotic members of the Bcl-2 family regulate mitochondrial activities relevant to apoptotic signaling by influencing the realaseing of cyt c.
Apoptosis
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Caspases/metabolism*
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Cytochrome c Group/metabolism*
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Humans
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Hypoxia, Brain/pathology*
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Membrane Proteins/metabolism*
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Mitochondria/metabolism*
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Neurons/pathology*
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Proto-Oncogene Proteins c-bcl-2/metabolism*
;
Signal Transduction
3.The cytosolic domain of Bcl-2 forms small pores in model mitochondrial outer membrane after acidic pH-induced membrane association.
Jun PENG ; Suzanne M LAPOLLA ; Zhi ZHANG ; Jialing LIN
Journal of Biomedical Engineering 2009;26(1):130-137
The permeability of mitochondrial outer membrane (MOM) is regulated by the proteins of the Bcl-2 family via their interactions at the membrane. While pro-apoptotic Bax protein promotes MOM permeabilization (MOMP) releasing cytochrome c after activation by BH3-only protein, anti-apoptotic Bcl-2 protein protects MOM. However both Bax and Bcl-2 can form pores in model membranes. Unlike Bax pore that has been extensively studied and reported to be directly linked to MOMP, Bcl-2 pore is much less known; thus we investigated the pore-forming property of recombinant Bcl-2 lacking the C-terminal transmembrane sequence (Bcl-2deltaTM) in liposomal membranes of MOM lipids. We found that: (1) Bcl-2 formed pores at acidic pH that induced the association of Bcl-2 with liposome; (2) Bcl-2 pore size was dependent on Bcl-2 concentration, suggesting that oligomerization is involved in Bcl-2 pore formation; (3) Unlike Bax pore that could release large molecules up to 2 mega-Da, Bcl-2 pore was smaller and could only release the molecules of a few kilo-Da. Therefore, Bcl-2 and Bax may form different size pores in MOM, and while the large pore formed by Bax may release cytochrome c during apoptosis, the small pore formed by Bcl-2 may maintain the normal MOM permeability.
BH3 Interacting Domain Death Agonist Protein
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metabolism
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Cell Membrane Permeability
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Cytochrome c Group
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metabolism
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Humans
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Hydrogen-Ion Concentration
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Liposomes
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metabolism
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Mitochondrial Membrane Transport Proteins
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metabolism
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Mitochondrial Membranes
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metabolism
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bcl-2-Associated X Protein
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metabolism
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bcl-X Protein
;
metabolism
4.Relationship between cytochrome c-mediated caspase-3 activity and chemoresistance in cisplatin-resistant human ovarian cancer cell lines.
Xiaokui YANG ; Fang ZHENG ; Hui XING ; Qinglei GAO ; Wei WANG ; Yunping LU ; Shixuan WANG ; Ding MA
Chinese Journal of Oncology 2002;24(6):544-547
OBJECTIVETo investigate the effects of anti-apoptosis gene (bcl-X(L)), cytochrome c and caspase-3 activity on chemoresistance in cisplatin-resistant human ovarian cancer cell lines (A2780/DDP, COC1/DDP).
METHODSThe expression of bcl-X(L) cisplatin treated cytochrome c and caspase-3 activity were monitored by RT-PCR and Western blot in cisplatin-resistant (A2780/DDP, COC1/DDP) and cisplatin-sensitive (A2780, COC1) cell lines. The apoptotic rates of A2780, COC1, A2780/DDP and COC1/DDP were detected with flow cytometry after having been treated by cisplatin.
RESULTSThe expression of bcl-X(L) in A2780/DDP and COC1/DDP was significantly higher than that in A2780 and COC1 cells, whereas the expression of cytochrome c, caspase-3 activity and apoptotic rates of A2780/DDP and COC1/DDP were significantly reduced more than those of A2780 and COC1 after having been treated by cisplatin (P < 0.05).
CONCLUSIONThe overexpression of anti-apoptotic gene bcl-X(L), which downregulates cytochrome c and decreases caspase-3 activity, may be related to cisplatin-resistance in human ovarian cancer cell lines.
Antineoplastic Agents ; pharmacology ; Apoptosis ; Caspase 3 ; Caspases ; metabolism ; Cisplatin ; pharmacology ; Cytochrome c Group ; metabolism ; Drug Resistance, Multiple ; physiology ; Drug Resistance, Neoplasm ; physiology ; Female ; Humans ; Ovarian Neoplasms ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Tumor Cells, Cultured ; bcl-X Protein
5.Modulatory effect of fumaric acid esters on superoxide-anion generation in human phagocytes.
Ke-jian ZHU ; Jian-ping CEN ; Ai-hua LIN ; Na JIN ; Hao CHENG
Acta Pharmaceutica Sinica 2007;42(11):1215-1217
Fumaric acid esters (FAE), mainly dimethylfumarate (DMF), have been shown to be highly efficacious in the treatment of psoriasis. Among the potential side effects of FAE therapy, lymphocytopenia is sometimes observed. In order to address the question whether FAE may interfere with systems of the innate defense, the modulatory role of FAE on the generation of superoxide-anion by human monocytes and neutrophils was studied by measuring the reduction of cytochrome c. Various concentrations of DMF and its metabolite methylhydrogenfumarate (MHF) were used to observe their modulatory effect on superoxide-anion generation by monocytes and neutrophils in response to bacteria (S. aureus and E. coli) and candida (C. albicans). Dexamethasone (DXM, 1 x 10(-7) mol x L(-1)) was also studied at the same time. We found that DXM significantly inhibited superoxide-anion generation from monocytes in response to bacteria and C. albicans, whereas DMF and MHF (10-20 microg x mL(-1)) significantly increased the production of superoxide-anion in monocytes in response to the above mentioned bacteria. DXM, DMF and MHF did not affect superoxide-anion generation of neutrophils. Our data indicate that DMF and MHF enhance superoxide-anion generation in human monocytes as one of the important mechanisms of innate defense against microorganisms.
Candida albicans
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immunology
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Cells, Cultured
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Cytochrome c Group
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metabolism
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Dermatologic Agents
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pharmacology
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Dimethyl Fumarate
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Escherichia coli
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immunology
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Fumarates
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pharmacology
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Humans
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Phagocytes
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metabolism
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Staphylococcus aureus
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immunology
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Superoxides
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metabolism
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Zymosan
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immunology
6.Herba houttuyniae extract induces apoptotic death of human promyelocytic leukemia cells via caspase activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release.
Kang Beom KWON ; Eun Kyung KIM ; Byung Cheul SHIN ; Eun A SEO ; Jeong Yeh YANG ; Do Gon RYU
Experimental & Molecular Medicine 2003;35(2):91-97
Herba houttuyniae has been used as a constituent of herval medicine prescriptions for the treatment of inflammation, cancer, and other diseases. In the present study, we investigated the cellular effects of herba houttuyniae extract (HHE) and the signal pathways of HHE-induced apoptosis in HL-60 human promyelocytic leukemia cell line. HHE treatment caused apoptosis of cells as evidenced by discontinuous fragmentation of DNA, the loss of mitochondrial membrane potential, release of mitochondrial cytochrome c into the cytosol, activation of procaspase-9 and caspase-3, and proteolytic cleavage of poly(ADP-ribose) polymerase. Pretreatment of Ac-DEVD-CHO, caspase-3 specific inhibitor, or cyclosporin A, a mitochondrial permeability transition inhibitor, completely abolished HHE-induced DNA fragmentation. Together, these results suggest that HHE possibly causes mitochondrial damage leading to cytochrome c release into cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic cell death in HL-60 cells.
Apoptosis/*drug effects
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Caspases/*metabolism
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Cytochrome c Group/*metabolism
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Enzyme Activation/drug effects
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HL-60 Cells
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Human
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Medicine, Oriental Traditional
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Membrane Potentials/drug effects
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Mitochondria/*drug effects
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Plant Extracts/*pharmacology
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Plants, Medicinal/*chemistry
7.Activation of the intrinsic mitochondrial apoptotic pathway in swine influenza virus-mediated cell death.
Young Ki CHOI ; Tae Kyung KIM ; Chul Joong KIM ; Joong Seob LEE ; Se Young OH ; Han Soo JOO ; Douglas N FOSTER ; Ki Chang HONG ; Seungkwon YOU ; Hyunggee KIM
Experimental & Molecular Medicine 2006;38(1):11-17
The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK-stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.
Animals
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Annexin A5/metabolism
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*Apoptosis
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Blotting, Western
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Cell Fractionation
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Cell Line
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Comparative Study
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Cytochrome c Group/metabolism
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Cytosol/chemistry
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DNA Fragmentation
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Enzyme Activation
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Gene Expression Regulation, Viral
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Hela Cells
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Humans
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Influenza A virus/*physiology
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Kinetics
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Mitochondria/metabolism/*physiology
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Precipitin Tests
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Proto-Oncogene Proteins c-bcl-2/genetics/metabolism
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Research Support, Non-U.S. Gov't
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Swine
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bcl-2-Associated X Protein/genetics/metabolism
8.Antitumor effect of capsaicin on colorectal carcinoma xenograft in nude mice.
Li-li ZHU ; Wan-le HU ; Lin-jun ZHANG ; Zhi-gao YU ; Chong-jie HUANG ; Ming-zhe JIANG ; Ming-xing TENG ; Jian-lu LIU ; Chang-bao LIU
Chinese Journal of Oncology 2013;35(4):256-261
OBJECTIVETo evaluate the effect of capsaicin on nude mice xenografted with colorectal carcinoma cells, and to explore its mechanism of action.
METHODSA nude mouse model of colorectal cancer was established by subcutaneous inoculation of human colorectal carcinoma HT-29 cells. Terminal deoxynucleotidyl transferase-mediated nicked labeling assay (TUNEL) was undertaken to detect the cell proliferation and apoptosis in the xenograft tissue in nude mice. Immunohistochemical (IHC) staining and Western blot were used to detect the expression of HSP27, Cyt-C and active caspase-3.
RESULTSThe tumor growth of the groups C10 and C20 was significantly slower than that of the group NS. The integrated optical density (IOD) of both the group C5 (2532.14 ± 578.11) and group C10 (6364.03 ± 1137.98) was significantly higher than that of the group NS (760.12 ± 238.05), (P < 0.05). The integrated optical density (IOD) of the group C20 was (15743.96 ± 1855.95), significantly higher than that of the groups C10, C5 and NS (all were P < 0.01). Immunohistochemistry showed that the cytoplasmic expression of HSP27 was strongly positive in the group NS, and significantly reduced with the increasing dose of capsaicin in the treated groups. The expression of active caspase-3 and Cyt-C in the group NS was weakly positive, and was significantly increased with the increasing dose of capsaicin in the groups C5 and C10 (P < 0.05), and the expression of active caspase-3 and Cyt-C of the group C20 was significantly higher than that of the groups C5, C10 and NS (P < 0.01). Western blot analysis showed that both the expressions of HSP27 of the group C5 (0.73 ± 0.05) and the group C10 (0.41 ± 0.03) were significantly lower than that of the group NS (P < 0.05). The expression of HSP27 of the group C20 (0.22 ± 0.06) was significantly lower than that of the groups C5, C10 and NS (P < 0.01). The expressions of active-caspase-3 and Cyt-C in the group C5 were (2.57 ± 0.34) and (2.03 ± 0.38), significantly higher than those of the group NS (P < 0.05). The expressions of active-caspase-3 and Cyt-C in the group C10 were (4.23 ± 0.45) and (3.13 ± 0.44), also significantly higher than those of the group NS (P < 0.05). The expressions of active-caspase-3 and Cyt-C in the group C20 were (5.78 ± 0.48) and (4.92 ± 0.52), significantly higher than those of the group C5, C10 and NS (P < 0.01). TUNEL analysis showed that there was a significant difference of cell apoptosis in comparison of each two groups. The higher dose of capsaicin was used, the more apoptosis was observed.
CONCLUSIONSCapsaicin can significantly inhibit the tumor growth and induce cell apoptosis in the colorectal carcinoma xenograft in nude mice. Its mechanism of action is possibly related with the down-regulation of HSP27 expression and up-regulation of expression of active caspase-3 and Cyt-C in the colorectal carcinoma xenograft in nude mice.
Animals ; Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Capsaicin ; administration & dosage ; pharmacology ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Cytochrome c Group ; metabolism ; Dose-Response Relationship, Drug ; Female ; HSP27 Heat-Shock Proteins ; metabolism ; HT29 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Random Allocation ; Tumor Burden ; Xenograft Model Antitumor Assays
9.The mechanism of STI571 inducing apoptosis of K562 cells.
Yazhen QIN ; Shanshan CHEN ; Yan CHANG ; Jiayu FU ; Xinjuan WANG
Chinese Journal of Hematology 2002;23(6):289-292
OBJECTIVETo investigate the mechanism of STI571 inducing apoptosis of K562 cells which express P210(BCR/ABL).
METHODSApoptosis was analyzed by Annexin-V/PI, DioC6 [3] staining, DCFH-DA staining, DNA-PI staining and DNA ladder. Western blot was used to analyse mitochondrial and cytosolic cyto C, Bcl-X(L), caspase-3, actin protein and the level of tyrosine phosphorylation.
RESULTSAfter exposure to STI571, K562 cells were induced to apoptosis. Tyrosine phosphorylation level of P210(BCR/ABL) and Bcl-X(L) was decreased. Caspase-3 was activated and there was an cytosolic accumulation of cyto C.
CONCLUSIONSTI571 could rapidly decrease the tyrosine phosphorylation level of P210(BCR/ABL). The signal pathway mediated by the cytosolic translocation of mitochondrial cyto C was one of the mechanisms that STI571 inducing apoptosis. STI571 was an effective gene targeting therapeutic agent.
Antineoplastic Agents ; pharmacology ; Apoptosis ; Benzamides ; Caspase 3 ; Caspases ; metabolism ; Cytochrome c Group ; metabolism ; Cytoplasm ; metabolism ; Enzyme Precursors ; metabolism ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Imatinib Mesylate ; K562 Cells ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology ; Reactive Oxygen Species ; metabolism
10.Human Telomerase Reverse Transcriptase (hTERT): A Target Molecule for the Treatment of Cisplatin-resistant Tumors.
Yuk Pheel PARK ; Kwang Dong KIM ; Seong Ho KANG ; Do Young YOON ; Joo Won PARK ; Jong Wan KIM ; Hee Gu LEE
The Korean Journal of Laboratory Medicine 2008;28(6):430-437
BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS: To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS: hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS: These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.
Amino Acid Chloromethyl Ketones/pharmacology
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Antineoplastic Agents/*pharmacology
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Caspases/antagonists & inhibitors/metabolism
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Cell Line, Tumor
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Cisplatin/*pharmacology
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Cysteine Proteinase Inhibitors/pharmacology
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Cytochrome c Group/metabolism
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Drug Resistance, Neoplasm/genetics
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Humans
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Neoplasms/therapy
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RNA, Small Interfering
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Telomerase/*antagonists & inhibitors/genetics/metabolism
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bcl-2-Associated X Protein/metabolism